عزل وتجنيس فايروس مرض نيوكاسل محليا من قطعان الدواجن المصابة طبيعيا في شمال العراق == ISOLATION AND GENOTYPING OF LOCAL NEWCASTLE DISEASE VIRUS ISOLATED FROM NATURALLY INFECTED CHICKENS IN NORTHERN IRA
Author name:
احمد ابراهيم احمد
Supervisor name:
شوني ميخائيل اوديشو | رباح نجاح جبار
General topic:
Veterinary Medicine
Specific topic:
Microbiology
Degree:
Doctorate
University:
University of Baghdad - College Of Veterinary Medicine
Language:
English
University location:
Baghdad
First pages:
21T550 - p.pdf
Abstract:
Newcastle disease (ND) has been concerned as one of the most important devastating diseases of poultry because of its wide range host of birds and worldwide distribution with severe economic losses in domestic poultry, due to the severe nature of the disease, ND is included as a list A disease.
Iraq has been endemic for Newcastle disease virus (NDV) with natural infection causing significant losses in the poultry industry since 1968 by the NDV/AG68 strain (AF001108) from infected chickens at Abu Graib in Iraq.
Currently, NDV genotype VII are most frequently associated with outbreaks of ND in the Middle East and Asia which have showed higher mortality in vaccinated poultry. The main objective of this study was to determine the biological and molecular characterization of the pathogenicity and genotyping followed by phylogenetic analysis. To evaluating the degree of genetic diversity of local isolated NDV strains and estimating the relationships to that NDV presence in the neighboring region, as well as preparation of inactivated vaccine against NDV from local strains was determine.
At the beginning, this study was carried out to isolate Newcastle disease virus (NDV) in naturally infected chickens of poultry farms in the north of Iraq. The study initiated diagnosis of 46 farms out of 96 broiler farms exhibited Newcastle disease (ND) (26 in Erbil, 14 in Sulaymanhia and 6 in Duhok) based on clinical signs corresponding with (8-15%) mortality and gross lesions of scarified chickens. The rapid test for detection NDV antigen from infected chickens for tracheal swab indicated that 32 farm samples were positive out of 46 initiative diagnosed farms.
Based on intiative diagnosis the identification of local isolates of NDV samples was pooled lung with trachea, spleen, cecal tonsil and intestine, were pooled in nine samples, then tissue homogenates were made from all samples in sterile Phosphate Buffered Saline (PBS) containing antibiotics, after centrifugation of tissue homogenates the supernatant fluids were assessed to real time reverse transcriptase- Polymerase Chain Reaction (RRT-PCR) assay. The results indicated that six
homogenate samples were positive for NDV and negative for Avian Influenza Virus.
After that, the remain supernatant fluids were used for virus isolation in embryonated chicken eggs then harvested allantoic fluids were used for employed for the hemagglutination activity. Six samples were showed positive results. Furthermore, virus identification was also performed by using conventional diagnostic methods such as Hemagglutination(HA) of red blood cells followed by Hemagglutination Inhibition (HI) test applying the prepared hyper immune serum(HIS) against NDV antigens to identify the NDV. The serological results demonstrated that six samples of NDV were positive, as well as the (rt-PCR) assay was performed for allantoic fluid to insure the presence of NDV using specific primers and probes to amplify specific region in the M gene of the viruses. The results revealed that six isolates were positive for NDV.
In addition, the pathogenicity of the six isolates was performed according to standard methods including Mean death times (MDT), Intra-cerebral pathogenicity index (ICPI) and Intra-venous pathogenicity index(IVPI), which revealed that six isolates were compatible to mesogenic (1/6) and velogenic (5/6) types.
At the second step, the molecular characterization of six isolates were investigated in Borehingher laboratory (Germany). The RT-PCR successfully amplified a 436 bp DNA fragment covering a part of the matrix (M) gene, fusion (F) gene (lentogenic) and fusion (F) gene (subgenotype VIIi) from all six isolates (NDV). The result was indicated that 5/6 were positive for velogenic NDV strain of genotype VIIi and 1/6 were negative. The nucleotide sequences of F gene were also determined, where the portion of the F2 gene of five isolates compared with that of other NDV strains retrieved from GenBank. Phylogenetic analysis showed that the isolates NDV belong to genotypes VIIi.
However, in local laboratory the result of FAST one-step quantitative Polymerase Chain Reaction (qPCR) successfully amplified a 525 bp fragment covering a part of the fusion (F1) gene and 515 bp fragment covering a part of the fusion (F2) gene from all six isolates. The nucleotide sequences of the PCR product of E1, S1 and S2
were found in F1 gene, while E1, E2, S1, S2, and D2 found in F2 gene out of six NDV isolates. Those results were compared with other NDV strains which retrieved from GenBank. Phylogenetic analysis showed that the Iraqi isolates are belonging to genotypes VII.
Finally, Inactivated vaccine was prepared locally using Velogenic NDV genotype VIIi isolate as a seed virus ELD50/0.1ml109 then compared with a commercial inactivated vaccine in an experimental study. This study was conducted by purchasing 120 broiler chicks and divided into three equal groups. Group 1 as unvaccinated control group , G2 for commercial vaccine and G3 for local vaccine), G2 and G3 were injected s/c with inactivated vaccine at 3 days old followed by a booster dose with LaSota live vaccine eye drop. Indirect ELISA technique was used to measure of antibody titer at ages 7, 17 and 27 days old (pre-challenge), the result showed at 7 days-old were (4251±1078) for all groups, and at 17 days-old were (906±33, 844±153 and 1145±219) for G1, G2 and G3 respectively. There were significant differences (p
Iraq has been endemic for Newcastle disease virus (NDV) with natural infection causing significant losses in the poultry industry since 1968 by the NDV/AG68 strain (AF001108) from infected chickens at Abu Graib in Iraq.
Currently, NDV genotype VII are most frequently associated with outbreaks of ND in the Middle East and Asia which have showed higher mortality in vaccinated poultry. The main objective of this study was to determine the biological and molecular characterization of the pathogenicity and genotyping followed by phylogenetic analysis. To evaluating the degree of genetic diversity of local isolated NDV strains and estimating the relationships to that NDV presence in the neighboring region, as well as preparation of inactivated vaccine against NDV from local strains was determine.
At the beginning, this study was carried out to isolate Newcastle disease virus (NDV) in naturally infected chickens of poultry farms in the north of Iraq. The study initiated diagnosis of 46 farms out of 96 broiler farms exhibited Newcastle disease (ND) (26 in Erbil, 14 in Sulaymanhia and 6 in Duhok) based on clinical signs corresponding with (8-15%) mortality and gross lesions of scarified chickens. The rapid test for detection NDV antigen from infected chickens for tracheal swab indicated that 32 farm samples were positive out of 46 initiative diagnosed farms.
Based on intiative diagnosis the identification of local isolates of NDV samples was pooled lung with trachea, spleen, cecal tonsil and intestine, were pooled in nine samples, then tissue homogenates were made from all samples in sterile Phosphate Buffered Saline (PBS) containing antibiotics, after centrifugation of tissue homogenates the supernatant fluids were assessed to real time reverse transcriptase- Polymerase Chain Reaction (RRT-PCR) assay. The results indicated that six
homogenate samples were positive for NDV and negative for Avian Influenza Virus.
After that, the remain supernatant fluids were used for virus isolation in embryonated chicken eggs then harvested allantoic fluids were used for employed for the hemagglutination activity. Six samples were showed positive results. Furthermore, virus identification was also performed by using conventional diagnostic methods such as Hemagglutination(HA) of red blood cells followed by Hemagglutination Inhibition (HI) test applying the prepared hyper immune serum(HIS) against NDV antigens to identify the NDV. The serological results demonstrated that six samples of NDV were positive, as well as the (rt-PCR) assay was performed for allantoic fluid to insure the presence of NDV using specific primers and probes to amplify specific region in the M gene of the viruses. The results revealed that six isolates were positive for NDV.
In addition, the pathogenicity of the six isolates was performed according to standard methods including Mean death times (MDT), Intra-cerebral pathogenicity index (ICPI) and Intra-venous pathogenicity index(IVPI), which revealed that six isolates were compatible to mesogenic (1/6) and velogenic (5/6) types.
At the second step, the molecular characterization of six isolates were investigated in Borehingher laboratory (Germany). The RT-PCR successfully amplified a 436 bp DNA fragment covering a part of the matrix (M) gene, fusion (F) gene (lentogenic) and fusion (F) gene (subgenotype VIIi) from all six isolates (NDV). The result was indicated that 5/6 were positive for velogenic NDV strain of genotype VIIi and 1/6 were negative. The nucleotide sequences of F gene were also determined, where the portion of the F2 gene of five isolates compared with that of other NDV strains retrieved from GenBank. Phylogenetic analysis showed that the isolates NDV belong to genotypes VIIi.
However, in local laboratory the result of FAST one-step quantitative Polymerase Chain Reaction (qPCR) successfully amplified a 525 bp fragment covering a part of the fusion (F1) gene and 515 bp fragment covering a part of the fusion (F2) gene from all six isolates. The nucleotide sequences of the PCR product of E1, S1 and S2
were found in F1 gene, while E1, E2, S1, S2, and D2 found in F2 gene out of six NDV isolates. Those results were compared with other NDV strains which retrieved from GenBank. Phylogenetic analysis showed that the Iraqi isolates are belonging to genotypes VII.
Finally, Inactivated vaccine was prepared locally using Velogenic NDV genotype VIIi isolate as a seed virus ELD50/0.1ml109 then compared with a commercial inactivated vaccine in an experimental study. This study was conducted by purchasing 120 broiler chicks and divided into three equal groups. Group 1 as unvaccinated control group , G2 for commercial vaccine and G3 for local vaccine), G2 and G3 were injected s/c with inactivated vaccine at 3 days old followed by a booster dose with LaSota live vaccine eye drop. Indirect ELISA technique was used to measure of antibody titer at ages 7, 17 and 27 days old (pre-challenge), the result showed at 7 days-old were (4251±1078) for all groups, and at 17 days-old were (906±33, 844±153 and 1145±219) for G1, G2 and G3 respectively. There were significant differences (p
Summary:
b419cbdd444c58c33f97d6c563877992.pdf
References:
592647eac6632d50b96986ead36f6fae.pdf
